Boiling histotripsy exhibits anti-fibrotic effects in animal models of liver fibrosis

Liver fibrosis is a hallmark of chronic liver disease which could lead to liver cirrhosis or liver cancer. However, there is currently lack of a direct treatment for liver fibrosis. Boiling histotripsy (BH) is an emerging non-invasive high-intensity focused ultrasound technique that can be employed to mechanically destruct solid tumour at the focus via acoustic cavitation without significant adverse effect on surrounding tissue. Here, we investigated whether BH can mechanically fractionate liver fibrotic tissue thereby exhibiting an anti-fibrotic effect in an animal model of liver fibrosis. BH-treated penumbra and its identical lobe showed reduced liver fibrosis, accompanied by increased hepatocyte specific marker expression, compared to the BH-untreated lobe. Furthermore, BH treatment improved serological liver function markers without notable adverse effects. The ability of BH to reduce fibrosis and promote liver regeneration in liver fibrotic tissue suggests that BH could potentially be an effective and reliable therapeutic approach against liver fibrosis.


BH attenuates thioacetamide (TAA)-induced liver fibrosis
A fibrotic liver is generally stiffer than a healthy normal liver.In the present study, BH treatment was successfully performed to mechanically fractionate fibrotic liver tissue in vivo (Fig. 1A to F) with the BH protocol previously used for normal liver fractionation 16 .The morphological chronological changes of the liver after the BH treatment are shown in Fig. 1B.In the acute phase, 7 days after the BH treatment, both the BH-treated core and penumbra showed relatively higher liver fibrosis scores than the BH-untreated lobe, although the difference was not statistically significant.There was also no significant difference between the BH-treated identical lobe and the untreated lobe (Fig. 2A to F, and S).In contrast, 21 and 90 days after the BH treatment, both the BH-treated penumbra and BH-treated identical lobe showed significantly reduced liver fibrosis scores compared to the BHuntreated lobe.Furthermore, the BH-treated core also exhibited a lower fibrosis score than the BH-untreated lobe (Fig. 2G to R, and S).Both α-smooth muscle-actin (α-SMA) and vimentin are well-known molecular markers for liver fibrosis 17,18 .On the 21st day after the BH treatment, all the BH-treated lobes showed reduced α-SMA expression compared to the BH-untreated lobe.90 days after BH treatment, both the BH-treated penumbra and the BH-treated identical lobes exhibited reduced α-SMA expression compared to the BH-untreated lobe.Furthermore, the α-SMA expression of all the BH-treated lobes was not significantly different from the sham group.However, the BH-untreated lobe still showed a significant upregulation of α-SMA expression compared to the sham liver (Fig. 3A to I, and J).As shown in Fig. 3K, the expression of vimentin was upregulated in all TAA-induced fibrotic livers at 21 days after BH treatment, whereas the BH-treated penumbra exhibited reduced vimentin expression compared to the BH-untreated lobe at 90 days after the BH treatment.
Next, we evaluated the effect of BH on collagen content in TAA-induced liver fibrosis.After 21 days of the BH treatment, when compared with the BH-untreated lobe, the expression of collagen type I was reduced in both the BH-treated penumbra and the BH-treated identical lobe.Similarly, the expression of collagen type III was significantly decreased in the BH-treated penumbra.The total collagen content was also significantly decreased in the BH-treated penumbra and the BH-treated identical lobe (Fig. 4A to C).After 90 days of the BH treatment, the expression of collagen types I, III, and total collagen content was significantly reduced in the BH-treated penumbra compared to the BH-untreated lobe (Fig. 4A to C).These results indicate that BH treatment could reduce fibrotic tissues in a TAA-induced liver fibrosis animal model.

BH promotes liver regeneration
The expression of asialoglycoprotein receptor 1 (ASGR1), a hepatocyte specific marker 19 , was significantly increased in the BH-treated penumbra compared to the BH-untreated lobe at all 7, 21, and 90 days after the BH treatment (Fig. 3A to I, and L).Next, we assessed the expression of CD26, which is also well-known as a hepatocyte marker 20,21 .On the 7th day after the BH treatment, both BH-treated and BH-untreated lobe showed significantly reduced CD26 expression compared to the sham group (Fig. 5A to C, and J).21 days after the BH exposure, although both BH-treated and BH-untreated lobes still showed reduced CD26 expression compared to the sham, the BH-treated penumbra and BH-treated identical areas exhibited significantly increased CD26 expression compared to the BH-untreated lobe (Fig. 5D to F, and J).On the 90th day after the BH treatment, all BH-treated cores, BH-treated penumbras, and BH-treated identical lobes showed significantly increased CD26 expression compared to the BH-untreated lobe.Furthermore, the entire BH-treated lobe showed no statistically significant differences in CD26 expression compared to the sham, whereas the BH-untreated lobe still exhibited  B, E, H) The BH-unaffected region in the BH-treated LLL.(C, F, I) BH-untreated ML. (Scale bar, 1 mm).Images (c′), (p′), (i′), and (ii′) show the highlighted areas in (A to I, square with broken lines) at higher magnifications with three channels (Red, Green, and Blue).Images (c″), (p″), (i″), and (ii″) show the highlighted areas in (A to I, square with broken lines) at higher magnifications with two channels (Red and Green).(Scale bar, 250 µm; magnification ×100).c: BH-treated core, p: BH-treated penumbra, i: severe fibrotic region, ii: mild fibrotic region.

Effect of BH on liver inflammatory cells
We did not observe any ectopic infiltration of monocytes/macrophages one week after the BH treatment in the left lateral lobe (LLL) of the liver, compared with the basal CD68 expression level in non-fibrotic healthy liver tissue and in BH-untreated lobes with TAA-induced liver fibrosis (Fig. 5A to C, and K).On day 21 after the BH treatment, we observed a slight decrease in CD68 expression levels compared with the basal CD68 expression level, regardless of the BH-treated region (Fig. 5D to F, and K).At day 90, CD68 expression levels in the BHtreated lobe had recovered, and this recovery was more pronounced in distal regions compared to the core of BH treatment.In contrast, the decreased CD68 level in the BH-untreated lobe was not restored by day 90 (Fig. 5G to I, and K).At all 7, 21, and 90 days after the BH treatment, there were no statistically significant differences between the sham and the BH-treated or -untreated lobes.Thus, these results suggest that BH treatment on fibrotic liver tissue does not aggravate the inflammatory response.

Effect of BH on body weight and serologic liver function markers
In comparison to the group with TAA-induced liver cirrhosis and the group treated with BH but without liver cirrhosis, BH treatment for TAA-induced cirrhotic livers did not result in a statistically significant delay in body weight recovery (Fig. 6A).In addition, there was no statistically significant difference observed in the daily body weight reduction or recovery rate among the three groups (Fig. 6B).As shown in Fig. 6C and D, the BH treatment significantly reduced the TAA-induced elevation in serum AST and ALT levels.Furthermore, it also reduced serum bilirubin levels, which were increased in animal models with liver fibrosis (Fig. 6E).

Discussion
Uncontrolled liver fibrosis progresses to liver cirrhosis, which is a major contributor to liver failure and liver cancer 3,4 .Discovering approaches to attenuate liver fibrosis, thereby preventing the progression of liver cirrhosis or liver cancer, could have a significant clinical benefit.In this context, we have clearly demonstrated, for the first time, that BH treatment effectively reduces liver fibrosis and promotes liver regeneration in an animal model of liver fibrosis.This study clearly reported the long-term feasibility of inducing localised therapeutic effects by BH.The anti-fibrotic effect was highlighted in the BH-treated penumbra and the BH-treated identical lobe from 21 to 90 days after BH treatment on the fibrotic liver.Moreover, liver regeneration was also facilitated in the BHtreated penumbra from 7 to 90 days and in the BH-treated identical lobe from 21 to 90 days after BH treatment, respectively.Based on our results, BH treatment seems to destroy fibrotic tissue, thereby stimulating the proliferation of hepatocytes in both the BH-treated penumbra and the BH-treated identical lobe, resulting in an anti-fibrotic effect.
In this experimental study, the BH-treated core exhibited a higher liver fibrosis score (Fig. 2) and α-SMA expression (Fig. 3A) compared to both the BH-treated and BH-untreated lobes in the acute phase (7 days after BH treatment).This increase gradually decreased through days 21 and 90 after BH treatment.In line with these findings, our recent study on normal liver tissue revealed that the BH-treated core exhibited higher levels of α-SMA expression and collagen deposition compared to both the BH-treated penumbra and the BH-untreated area.These elevated levels gradually returned to normal values over the course of days 14 to 28, reflecting the normal healing process 16 .Consequently, the heightened fibrosis observed in the BH-treated core during the acute phase following BH treatment in the TAA-induced liver fibrosis animal model could be attributed to the combined effects of the normal healing process.
Accumulating evidence suggests that activated myofibroblasts, primarily hepatic stellate cells with α-SMA (+) and producers of collagen type I, play a key role in the progression of liver fibrosis. 20,22,23.Furthermore, the clearance of activated myofibroblasts is associated with the regression of liver fibrosis, leading to an emerging therapeutic target for novel treatment against liver fibrosis 20,22,23 .Indeed, the reduction of activated myofibroblast populations can attenuate the collagen fiber production, thereby reducing liver fibrosis.In this study, we found that BH treatment promoted the reduction of activated myofibroblasts in all BH-treated lobes compared to BHuntreated lobes at 21 and 90 days after BH treatment.Notably, α-SMA expression in BH-treated lobes was nearly returned to normal values 90 days after BH treatment.
In terms of safety, we observed that BH-treatment did not exacerbate the inflammatory response in the treated fibrotic liver compared to the sham animal and showed no adverse side effects on the treated animals.Consistent with our results, the First-in-Human study (NCT03741088) of histotripsy treatment on multifocal liver tumours also reports no adverse clinical side effects 24 .A histotripsy device (Edison®, HistoSonics, USA) to treat patients with liver tumours has recently been approved by the US Food and Drug Administration (FDA) in Oct 2023 25 .
Liver transplantation is regarded as a cure of both cancer and cirrhosis 26,27 .However, patients on the waiting list for liver transplantation are often delayed or delisted due to a shortage of available liver donors and deteriorated liver function beyond the accepted criteria for transplantation 26,27 .Thus, currently, to maximize the clinical benefit of liver transplantation, interventional therapy called Bridging therapy is widely performed to prevent patients from advancing beyond the specified criteria while awaiting an organ 27,28 .In this perspective, BH treatment could serve as a promising modality for Bridging Therapy, given its ability to inhibit liver fibrosis and promote liver regeneration.
Taken together, this study highlights the first in vivo investigation reporting the therapeutic efficacy of BH on animal models with fibrotic livers.BH treatment showed an excellent anti-fibrotic effect without any adverse side effects in fibrotic liver tissues.Therefore, it could be a promising novel therapeutic modality for the treatment of liver fibrosis in the future.

Induction of liver fibrosis
Sprague-Dawley rats (Male, 6 weeks old, body weight of 260 to 280 g) were from Koatech (Pyeongtaek, Republic of Korea) and kept under a 12-h light/dark cycle, with unrestricted access to water and food.Following a oneweek period of adaptation, rats were subjected to intraperitoneal injections of either a vehicle solution (0.9% normal saline) or TAA (300 mg/kg, 12 mg/mL in normal saline) three times a week for 28 days.TAA is an organosulfur compound well-known for inducing hepatic fibrosis and is regarded as an ideal fibrosis-inducing agent to evaluate the anti-fibrotic effect in experimental animals 29 .The Institutional Animal Care and Use Committee (IACUC) at Korea University granted approval for all experimental procedures and protocols under the reference number KOREA-2022-0120.All the methods were performed in accordance with the relevant guidelines and regulations.

Surgery for BH experiment
After 28 days of TAA administration, surgery for BH experiment was performed as previously mentioned 16 .
Anaesthesia induction was performed by administering 3.5% isoflurane within a ventilated anaesthesia chamber, utilizing a mixture of nitrous oxide and oxygen.Maintenance of anaesthesia was achieved through administering 2 to 2.5% isoflurane through a nasal cone in a 2:1 mixture of nitrous oxide and oxygen via inhalation.Next, the rats were situated on a heated pad, and a midline xipho-pubic laparotomy was executed to unveil the LLL of the liver.After BH treatment, the incisional site was sutured, and the subjects were permitted unrestricted access to food and water upon awakening.The sham animals only underwent xipho-pubic laparotomy with wound closure, without TAA administration and BH treatment.

BH experimental protocol
The same BH experimental protocol used in our previous study 16 was adopted (Fig. 1A), which successfully demonstrated its efficacy and long-term safety on normal liver tissue in vivo 16 .A 2 MHz-HIFU transducer (H148, Sonic Concepts, Bothwell, WA, USA), which was triggered by a function generator (33600A, Keysight Technologies, Santa Rosa, CA, USA) in conjunction with a power amplifier (1040L, Electronics & Innovation, Rochester, NY, USA), was employed to generate a number of BH lesions in the fibrotic liver.A laser pointer, affixed to the translation stage and aligned with the central axis of the HIFU source, was used for the guidance of the BH treatment onto the target surface of the exposed LLL of the liver.During our experiments, the HIFU focus was kept constant as 5 mm below the surface of the liver.Ten 2 MHz HIFU pulses each comprising a 10 ms pulse duration, P + of 89.1 MPa, P − of −14.6 MPa with 1% DC and 1 Hz PRF were used to produce a single BH lesion at a given target position in the fibrotic liver.BH lesions were generated using a raster-scanning approach.
The HIFU focus was systemically traversed across an area of 1.44 cm 2 (1.2 cm × 1.2 cm) in both the transverse and lateral directions with 2 mm step increments.

Histopathology
Rats were euthanised 7, 21, and 90 days after BH treatment by carbon dioxide inhalation.The regions to determine the fibrosis score for BH-treated groups on the liver tissue (LLL) were carefully selected as previously described 16 .The BH-treated core was chosen as the region treated with the BH.The BH-treated penumbra was selected from the same histological slides surrounding the BH-treated core.The top right part of the left lateral lobe (LLL) and the center of the median lobe (ML) were selected for the BH-untreated identical lobe (LLL) and BH-untreated (ML), respectively.Next, the liver tissues were fixed for 48 h in a 4% paraformaldehyde solution, followed by desiccation in 70% ethanol.Subsequently, the tissues were embedded in a paraffin block.Next, blocks were sectioned to a thickness of 4.5 μm and subsequently subjected to staining procedures utilising haematoxylin and eosin (H&E) as well as Masson's trichrome (MT) staining for histological examination.All stained images were assessed using the Zeiss Axio Scan Z1 (Carl Zeiss, Jena, Germany).The degree of fibrosis was also quantified using the Ishak scoring system 30 .Vol In the present study, we used ASGR1, which is widely accepted as a hepatocyte-specific marker in the field of hepatology 19 .Furthermore, ASGR1 can serve as a specific target molecule for the selective delivery of drugs or small molecules into hepatocytes 31 .In this context, we believe that ASGR1 is significant in representing the restoration of hepatocyte population in fibrotic liver.

Hydroxyproline assay
The total collagen content in the liver was quantified using a colorimetric hydroxyproline assay kit (ab222941, Abcam, Cambridge, MA, USA).Liver tissues were denatured with a papain solution, as previously described 33 , and the hydroxyproline assay was performed according to the manufacturer's instructions.

Blood analysis
The quantification of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and direct bilirubin was performed utilizing the FUJI DRI-Chemiclinical Chemistry Analyzer (FUJI DRI-CHEM 4000i, Fuji Film, Tokyo, Japan).

Statistical analysis
All data were expressed in terms of the mean and standard deviation.The normal distribution was assessed using the Shapiro-Wilk test.Parametric analysis employed a one-way analysis of variance (ANOVA) with subsequent post-hoc correction using Tukey's test, whereas non-parametric analysis was conducted through the Kruskal-Wallis test, followed by post-hoc testing utilizing the Conover test.Data analysis was conducted using MedCalc software version 18.5 (MedCalc Software Ltd, Ostend, Belgium) and SPSS software version 17.0 (SPSS Inc, Chicago, IL, USA).A P-value less than 0.05 was considered statistically significant.◂

Figure 1 .
Figure 1.(A) A schematic diagram of the experimental setup for boiling histotripsy (BH) treatment.HIFU; high intensity focused ultrasound.(B) Chronological gross morphological changes of both normal and fibrotic liver tissue on days 0, 7, 21 and 90 after the BH treatment.The square with broken line indicates BH-treated regions.(Scale bar, 5 mm).(C-D) Gross and cross-sectional images of fibrotic liver tissues after BH treatment on Day 0. (C) Top view of BH-treated fibrotic liver tissue after cardiac perfusion.The broken line indicates a cross-section line for histological observation.(Scale bar, 10 mm).(D) Cross section of BH-treated fibrotic liver tissue after cardiac perfusion.(Scale bar, 10 mm).(E-F) Histological images of (E) haematoxylin and eosin (H&E) or (F) Masson's trichrome-stained liver tissues collected on Day 0. (Scale bar, 1 mm).Images (i) show the highlighted areas (between BH-treated and untreated areas) in (E to F, enclosed by a square with broken lines) at higher magnifications.(Scale bar, 250 µm; magnification ×100).

Figure 2 .
Figure 2. Cross sectional images of the fibrotic liver tissues after BH treatment.(A-R) Histological images of (A-C, G-I, M-O) haematoxylin and eosin (H&E) or (D-F, J-L, P-R) Masson's trichrome stained liver tissues collected on days 7, 21, and 90 after the BH treatment.(A, D, G, J, M, P) BH-treated region in the left lateral lobe (LLL).(B, E, H, K, N, Q) The BH-unaffected region in the BH-treated LLL.(C, F, I, L, O, R) BH-untreated median lobe (ML).(Scale bar, 1 mm).Images (c), (p), and (i) show the highlighted areas in (A to R, square with broken lines) at higher magnifications.(Scale bar, 250 µm; magnification ×100).c: BH-treated core, p: BH-treated penumbra.(S) Liver fibrosis index score for each region of fibrotic liver samples at days 7, 21, and 90 after the BH treatment.n = 6 for sham, n = 6 for Day 7, n = 5 for Day 21, n = 3 for Day 90.All values are shown as means ± standard deviation (SD, # P < 0.05 vs. BH-untreated (ML) of each time point).

Figure 4 .
Figure 4. Collagen expression in fibrotic liver after BH treatment.(A) Transcriptome levels of collagen type I for each region of fibrotic liver at days 21, and 90 after the BH-treatment.n = 5 for sham, n = 6 for Day 21, n = 4 for Day 90.(B) Transcriptome levels of collagen type III for each region of fibrotic liver at days 21, and 90 after the BH-treatment.n = 6 for sham, n = 6 for Day 21, n = 4 for Day 90.(C) Quantification of the total collagen contents in each region of fibrotic liver at days 21 and 90 after the BH treatment.n = 6 for sham, n = 4 for Day 21, n = 4 for Day 90.All values are shown as means ± SD ( † P < 0.05 vs. Sham (LLL), † † P < 0.01 vs. Sham (LLL), † † † P < 0.001 vs. Sham (LLL), # P < 0.05 vs. BH-untreated (ML) of each time point, ## P < 0.01 vs. BH-untreated (ML) of each time point, ### P < 0.001 vs. BH-untreated (ML) of each time point).

Figure 6 .
Figure6.Recovery of surrogate markers of liver injury and body weight after TAA-induced liver fibrosis and BH treatment.(A) Changes in body weight after TAA-induced liver fibrosis and BH treatment.Week −4 to −1: n = 8 for BH-untreated fibrotic liver, n = 13 for BH-treated on fibrotic liver, Week 0 to 3: n = 9 for BH-treated on normal liver, n = 8 for BH-untreated fibrotic liver, n = 13 for BH-treated on fibrotic liver, Week 4 to 6: n = 9 for BH-treated on normal liver, n = 0 for BH-untreated fibrotic liver, n = 13 for BH-treated on fibrotic liver, Week 7 to 13: n = 9 for BH-treated on normal liver, n = 0 for BH-untreated fibrotic liver, n = 10 for BH-treated on fibrotic liver.Data are presented as means ± SD.One-way ANOVA followed by a post-hoc Tukey's test was used.No significant change was observed.(B) Changes in body weight differences during a week (ΔBody weight: Week n + 1 body weight − Week n body weight) after TAA-induced liver fibrosis and BH treatment.(C-E) Changes in serum AST, ALT, and direct bilirubin in response to TAA-induced liver fibrosis and BH treatment.Day 0 to 14: n = 4 for Sham, n = 8 for BH-untreated fibrotic liver, n = 24 for BH-treated on fibrotic liver, Day 21: n = 4 for Sham, n = 8 for BH-untreated fibrotic liver, n = 22 for BH-treated on fibrotic liver, Day 21: n = 4 for Sham, n = 8 for BH-untreated fibrotic liver, n = 22 for BH-treated on fibrotic liver, Day 28: n = 4 for Sham, n = 0 for BH-untreated fibrotic liver, n = 13 for BH-treated on fibrotic liver, Day 45 to 90: n = 4 for Sham, n = 0 for BH-untreated fibrotic liver, n = 6 for BH-treated on fibrotic liver.All values are shown as means ± SD ( † P < 0.05 vs. Sham, † † P < 0.01 vs. Sham, † † † P < 0.001 vs. Sham, # P < 0.05 vs. BH-untreated fibrotic liver, ### P < 0.001 vs. BH-untreated fibrotic liver).